No description available.

No description available.

No description available.

The 2,4-dinitrophenyl (DNP) group is an example of a hapten with potential use as a non-radioactive labelling group for oligonucleotides. This label is highly antigenic, inexpensive, chemically simple, sterically undemanding, and is not found <I>in vivo</I>. A series of non-nucleoside-based DNP phosphoramidites have been prepared and used in the multiple labelling of oligonucleotides during solid phase synthesis. Oligonucleotides labelled in this way were synthesised in very high yield and easily purified by reversed-phase HPLC. The lengths of spacer arms between the DNP label and the oligonucleotide phosphate backbone have been varied in order to determine the optimum length for anti-DNP antibody binding. The optimum number of DNP labels for maximum signal strength is also reported. The labelled oligonucleotides were detected using standard ELISA methodology, employing a monoclonal IgG mouse anti-DNP antibody, giving sensitivities equivalent to those obtainable in the visualisation of biotinylated oligonucleotides. DNP labelled phosphoramidite and triphosphate derivatives of 2'-deoxyuridine have also been synthesized and used to label oligonucleotides. As an alternative to the above DNP based non-radioactive labelling system, single and multiple dansyl based phosphoramidites have been synthesized and used to fluorescently label oligonucleotides. Protein-dansyl conjugates have been prepared for the production of anti-dansyl antibodies which would allow the immunogenic detection of this label. Oligonucleotide probes attached directly to alkaline phosphatase have been prepared using inexpensive, commercially available reagents and used in hybridisation experiments to detect 0.3fmoles of target DNA.

This thesis describes an investigation of the use of DNA junctions as functional nanoswitches, capable of molecular recognition tasks in nucleic acids. The topological branch point allows a conformational change from a planar, open structure to a closed conformation which allows the junction arms to stack. A stable nanoswitch was designed which formed a complete cruciform structure only when hybridised to a complementary target. Introduction of single base mismatches in the target at the nanoswitch branch point caused changes in FRET efficiency, depending upon the mismatch in question. This methodology was shown to be applicable to both DNA and RNA target sequences. The structural origins of these changes in FRET efficiency were then investigated using time-resolved fluorescence spectroscopy. Donor florescence decays were fitted using an assumption of a Gaussian distribution form for the fluorophore separation. The results showed a maximal change in peak position of 10 Angstroms, illustrating the sensitivity of these devices. The fluorescent analogue of adenine, 2-amino purine (2AP), was used as s direct probe of the branch point structure. Finally, a high throughput analysis mechanism was demonstrated. This used a fluorescence lifetime plate reader to obtain donor fluorescence decays for numerous samples in a short exposure time, which were used to determine the nanoswitch conformation. This method was initially shown to be applicable to DNA nanoswitches, with the use of FLIM also demonstrated, before the method was shown to be useful for distinguishing single base mismatches. Using a time-resolved approach has the benefit of being independent of local sample concentration, ideal for nanotechnology applications. These nanoswitches have been shown to be promising prototype molecular recognition devices.

The nucleic acids, RNA in particular, are conformationally complex biomolecules. The structures that they can form and the subtle sequence dependent modifications of conformation are of pivotal importance to their function <I>in vivo</I>. The advent of solid phase chemical synthesis methodologies for DNA and, more recently RNA, has fuelled the rapid growth in our knowledge of nucleic acid structure. The work described here is concerned with the development of rapid and reliable synthesis and purification protocols for the automated chemical synthesis of RNA. The use of RNA phosphoramidite monomers incorporating two different 2'-hydroxyl protecting groups is described, along with ion-exchange and reversed phase HPLC protocols for the purification of oligoribonucleotides. The quality of the material obtained by these methods has allowed detailed biophysical investigations by UV melting, high field NMR and X-ray crystallography.

No description available.

A new highly hydrophobic 5'-hydroxyl protecting group (Tbf-DMTr) has been designed for the purification of synthetic oligonucleotides. Tbf-DMTr-oligonucleotides are strongly retained on RP-HPLC allowing a facile separation from truncated sequences. Subsequently the group can be removed in acidic conditions. The fluorescent properties of TBf-DMTr enable easy detection. The synthesis and purification of long oligonucleotides (> 100-mer) has been undertaken. Additionally a new fluorescent label for oligonucleotides has been developed which enables detection of DNA probes to concentration down to 10-<SUP>10</SUP> mol/l.

Bacteriophage T3 has been reported to express S-adenosylmethionine hydrolase (SAMase) during the early stage of phage infection. In order to overcome the restriction-modification (R-M) system, SAMase destroys the modification cofactor S-adenosylmethionine (SAM) and hydrolyses SAM into homoserine and methylthioadenosine (MTA). Since SAM is also the major donor of the methyl groups incorporated in DNA methylation, this SAM cleaving activity may be utilized as a demethylating agent, which helps us to understand the mechanism of DNA demethylation. However, the reaction mechanism of T3 SAMase has not been very well studied. To further elucidate this mechanism, experiments were carried out to purify recombinant SAMase and to enable attempts to solve the crystal structure of this enzyme. In addition, we aimed to observe effects of DNA demethylation in mammalian cells by using this hydrolase activity to reduce the cellular level of SAM. SAMase was also chosen to substitute the more commonly used demethylating agent, 5-azaC, in order to avoid the highly toxic impact on drug-receiving cells. Additionally, RNA interference (RNAi), was utilised to explore the impact of DNA demethylation. Using small interfering RNA (siRNA), we depleted the mRNA of the enzyme responsible for maintenance methylation, DNA methyltransferase 1 (Dnmtl), and observed a correlation between DNA demethylation and <i>Xist </i>expression when the methyl-binding protein MBD2 was removed. Furthermore, the oxidative DNA repair has been suggested as a candidate pathway, which involves demethylation of the 1-methyladenine and 3-methylcytosine via a hydroxmethyl intermediate. To explore this possible mechanism, we investigated the candidate pathway by searching for a putative intermediate of hydroxymethyl cytosine during active DNA demethylation <i>in vivo.</i>

The cytoplasmic destination of transcripts is thought to be determined by the cis-acting sequences that usually lie in its 3’TR and act as zipcodes, by the trans-acting factors that interpret the localisation signals and by the type of motors that are engaged to transport RNP (Ribonucleoprotein) particles. The molecular motors Dynein and Kinesin transport their RNA cargoes over long distances towards the minus or plus ends of microtubule tracks respectively, whereas myosin mediates short-distance transport along actin microfilaments. Although many cargoes undergo net displacement and achieve asymmetric intracellular distribution over time, their motion is not always highly unidirectional and often interrupted by pauses and switches in direction. In Chapter 3 of this thesis, we investigate in detail the role of motors and their cofactors in bidirectional RNA motility in <i>Drosophila</i> embryos. We show that Kinesin I or Kinesin II are not involved in the reverse motion of dynein-driven RNA cargoes and we suggest that dynein moves bidirectionally <i>in vivo</i>. Furthermore, we demonstrate that dynactin, dynein’s accessory factor, is required to reduce backwards motility. We suggest that dynactin mediates suppression of reversals by stabilising dynein on the microtubule or by assembling and coordinating multiple dyneins together. In Chapter 4, we explore novel genome-wide bioinformatics approaches for the identification of localisation signals and localising transcripts in <i>Drosophila</i> embryos and oocytes and we show that the method successfully predicts localisation signals in transposable elements and in the 3’UTR of endogenous <i>Drosophila</i> genes. Finally, in Chapter 5, we address the suitability of various <i>Drosophila </i>cell lines for the establishment of an RNA localisation assay, with the aim of using the assay for a genome-wide RNAi screen for novel RNA localisation factors.